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N NMR Spin Relaxation Dispersion Study of the MolecularCrowding Effects on Protein Folding under Native Conditions

Xuanjun Ai, Zheng Zhou, Yawen Bai, and Wing-Yiu ChoyJ. Am. Chem. Soc. , 2006 , 128 (12), 3916-3917• DOI: 10.1021/ja057832n • Publication Date (Web): 02 March 2006

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15N NMR Spin Relaxation Dispersion Study of the Molecular Crowding Effectson Protein Folding under Native Conditions

Xuanjun Ai, † Zheng Zhou, ‡ Yawen Bai, ‡ and Wing-Yiu Choy* ,†

Department of Biochemistry, Uni Versity of Western Ontario, London, Ontario, Canada N6A 5C1, and NationalCancer Institute, Laboratory of Biochemistry, Building 37, Room 6114E, Bethesda, Maryland

Received November 17, 2005; E-mail: jchoy4@uwo.ca

In vitro studies of protein structure, dynamics, folding, and ligandbinding are usually performed using low concentrations of purifiedprotein in dilute buffer solutions. These environments, however,are dramatically different from that within cells where proteins carryout their functions. The presence of high concentrations of macromolecules, such as protein and RNA, occupy a significantfraction (typically 20 - 30%) of cellular volume and create acrowded environment inside a cell. 1- 6 The excluded volume effectbrought on by the high volume occupancy of solution by macro-molecules can affect the kinetics and thermodynamics of biochemi-

cal reactions by orders of magnitude. 1-

6 In particular, recent studieshave shown that the presence of high concentrations of macromol-ecules can significantly alter the stabilities and folding rates of proteins. 7- 10 This strongly suggests that the macromolecular crowd-ing effects may play a crucial role in promoting the folding of protein into functional form in cells.

Macromolecular crowding effects on protein folding processesare usually studied by conventional spectroscopic techniques suchas circular dichroism or fluorescence spectroscopy. Highly solubleand inert polymers such as dextran, poly(ethylene glycol) (PEG),and Ficoll are added to protein solutions to mimic the intracellularenvironments. 5 Even though these methods can be used to detectglobal structural changes of proteins under different environments,detailed structural and dynamical information at atomic resolution

cannot be obtained. Another disadvantage of using conventionalspectroscopic techniques to study the macromolecular crowdingeffects is that, on top of the crowding agents, a high concentrationof denaturant needs to be added to the sample to generate the proteinunfolded state. In this work, we demonstrated the feasibility of usingthe NMR relaxation dispersion technique to investigate the mac-romolecular crowding effects on protein folding under nativeconditions without involving any denaturant. The method has beenapplied to an engineered mutant of apocytochrome b562 .

Due to advancements in technology and methodology, NMR isa unique tool for protein dynamics studies. Protein motions over abroad range of time scales can now be probed by various spinrelaxation techniques. Recent studies demonstrated that proteinfolding transitions can also be studied using the newly developedrelaxation dispersion techniques. 11 These experiments measure thecontribution to transverse relaxation rates of nuclei from thechemical/conformational exchange process. Useful information suchas rates of exchange and the chemical shift differences betweenstates can be extracted from the relaxation dispersion profiles. In arecent work, we have used the 15N relaxation dispersion techniqueto extensively characterize the kinetics and thermodynamics of thefolding transitions of two mutational variants of a redesigned four-helix bundle protein (Rd-apocyt b562 ) under native conditions. 12

Temperature-dependent studies of the folding and unfolding rates

of the mutants has allowed thermodynamics parameters, includingenthalpy, entropy, and heat capacity of the proteins’ native andtransition states to be derived. This methodology provides a newand unique approach to obtain valuable kinetic and structuralinformation on protein folding under native conditions at atomiclevel.

To gain a better understanding of how protein stability andfolding are affected by the environment inside living cells, in thiswork, we extended the application of the 15N relaxation dispersiontechnique to the study of macromolecular crowding effects on

protein folding kinetics. Relaxation dispersion data of the F61A/ I72A mutant of Rd-apocyt b562 were collected at 600 and 800 MHz(1H frequency) as described in the previous study. 12 PEG 20K (MW≈ 20 000) was added to the protein sample to mimic the crowdedintracellular environment. To investigate the temperature depen-dence of the macromolecular crowding effects, dispersion profileswere also recorded for the protein at different temperatures (20,25 and 30 ° C). Relaxation dispersion data was then fitted to a two-state exchange model as established in our previous work. 12

Thermodynamics and kinetics of the folding transition process of Rd-apocyt b562 F61A/I72A in the presence and the absence of thecrowding agent were obtained. Figure 1 illustrates typical fits of dispersion profiles for a representative residue of the F61A/I72Amutant. The solid lines correspond to the best fits obtained fromglobal analysis of all residues at the two magnetic fields and atspecific temperatures. The parameters obtained from the two-state-folding model fitting of 15N relaxation dispersion data are sum-marized in Table 1. Note that data collected at different temperatureswere fitted individually. Our result clearly demonstrates that usefulinformation about macromolecular crowding effects on proteinfolding kinetics and thermodynamics can be extracted.

The 1H - 15N correlation spectrum of the protein obtained in thepresence of 85 mg/mL of PEG 20K is similar to the correspondingspectrum obtained without the crowding agent (Supporting Infor-mation), except that there are increases in peak line widths due tothe change in viscosity. This strongly suggests that the addition of crowding agent causes a minimal change to the protein structure,at least with the conditions used.

Significant increases in the protein folding rates, however, wereobserved at all three temperatures in the presence of a mildconcentration of crowding agent. At 20 ° C, the effect of macro-molecular crowding causes the folding rate ( k f ) of the protein toincrease by 80%, while the unfolding rate ( k u) remains unchangedwithin experimental error. As the temperature increases, theincreases in the folding rate due to the crowding effect seem tobecome less ( 33% increase at 30 ° C).

The changes in the folding rates of the protein indicate that theunfolded state of the protein is destabilized by macromolecularcrowding. This is reflected in the changes of the population of unfolded state ( pU ) and free energy difference ( ∆ G) between the

† University of Western Ontario.‡ National Cancer Institute.

Published on Web 03/02/2006

3916 9 J. AM. CHEM. SOC. 2006 , 128 , 3916 - 3917 10.1021/ja057832n CCC: $33.50 © 2006 American Chemical Society

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folded and unfolded states upon additions of PEG 20K. Table 1shows that the protein stability increases by 0.25 kcal/mol underthe crowding conditions. This result agrees quite well with whatQu and Bolen observed in their study of crowding effects on thestability of TCAM. 8 If the increase in stability of the F61A/I72Amutant is linearly proportional to the concentration of crowdingagent as observed for TCAM, 8 the stability of the F61A/I72Amutant will increase by 1.1 kcal/mol inside a cell, where theconcentration of macromolecules is approximately 400 mg/mL.Assuming that the unfolding rate of the protein is unaffected bythe crowding effects, the increase in stability will translate into a5.5-fold increase in the folding rate of the protein at 30 ° C.

Another advantage of using NMR relaxation dispersion tech-niques to study protein folding transitions is that structuralinformation about the protein unfolded state under native conditionscan be derived from the chemical shift difference between statesextracted from the relaxation dispersion profiles. Figure 2 showsthe 15N chemical shift differences ( ∆ ω ) between the folded stateand the unfolded state of F61A/I72A mutant of Rd-apocyt b562 inthe presence and the absence of crowding agent. The goodagreement of ∆ ω values under these two conditions stronglysuggests that the structure in the unfolded state is not perturbedsignificantly by the crowding agent, at least not at the concentrationused.

In summary, here we report a new and unique way to investigatethe macromolecular crowding effects on protein folding processesunder native conditions. By applying the 15N relaxation dispersiontechnique to an engineered mutant of Rd-apocyt b562 , the thermo-dynamics and kinetics parameters of the folding process, both inthe presence and the absence of crowding agent, can be extracted.

Studying the effects of crowding agent of different molecular sizesand at different concentrations on the thermodynamics and kineticsof the folding transition process will follow.

Acknowledgment. We thank Dr. Lewis E. Kay (University of Toronto) for the 800 MHz spectrometer time and many helpfuldiscussions. W.-Y.C. is a recipient of a Senior Research Fellowship(phase 2) from the Canadian Institutes of Health Research (CIHR).

This work was supported by a grant from CIHR.Supporting Information Available: Detailed materials and meth-

ods. This material is available free of charge via the Internet at http:// pubs.acs.org

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1993 , 22, 27 - 65.(2) Wenner, J. R.; Bloomfield, V. A. Biophys. J. 1999 , 77, 3234 - 3241.(3) Minton, A. P. Curr. Opin. Struct. Biol. 2000 , 10, 34 - 39.(4) Minton, A. P. J. Biol. Chem. 2001 , 276, 10577 - 10580.(5) Ellis, R. J. Trends Biochem. Sci. 2001 , 26, 597 - 604.(6) Sasahara, K.; McPhie, P.; Minton, A. P. J. Mol. Biol. 2003 , 326, 1227 -

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JA057832N

Figure 1. Typical fits of dispersion data at 800 MHz from a single residue of F61A/I72A mutant of Rd-apocyt b562 at (a) 20 ° C, (b) 25 ° C, and (c) 30 ° C,using a global two-state model in the absence (o) and in the presence (x) of 85 mg/mL of PEG 20K. R2

eff and VCPMG are the effective transverse relaxationrate and the CPMG field strength, respectively.

Table 1. Summary of Thermodynamics and Kinetics Parametersin the Presence and Absence of Crowding Agent a

temp (° C) k f (s- 1) k u (s- 1) pU (%) ∆ G (kcal/mol)

Without Crowding Agent20.0 1215 ( 67 8.9 ( 0.4 0.73 ( 0.02 - 2.86 ( 0.0225.0 1182 ( 34 13.9 ( 0.4 1.16 ( 0.02 - 2.63 ( 0.0130.0 1133 ( 15 28.0 ( 0.4 2.41 ( 0.02 - 2.22 ( 0.01

With 85 mg/mL PEG 20K20.0 2180 ( 253 10 ( 1 0.48 ( 0.02 - 3.10 ( 0.0325.0 1718 ( 107 13 ( 1 0.75 ( 0.02 - 2.89 ( 0.0230.0 1511 ( 36 25 ( 1 1.65 ( 0.04 - 2.46 ( 0.01

a k f folding rate , k u unfolding rate , pU population of the unfolded state,and ∆ G the free energy difference between the folded and unfolded state. Figure 2. 15N chemical shift differences between residues in the folded

state and unfolded state in the presence ( y-axis) and the absence ( x-axis) of PEG 20K.

C O M M U N I C A T I O N S

J. AM. CHEM. SOC. 9 VOL. 128, NO. 12, 2006 3917